Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni

The protein expression profiling in clam haemocytes and plasma in response to Perkinsus olseni was addressed. Adult Manila clams from a P. olseni‐free bed were experimentally challenged with parasite zoospores to analyse immune response. In another experiment, the effects of longer term infection were assessed in adult clams collected from a P. olseni‐affected bed, by comparing moderate to very heavily infected clams with non‐infected ones. Haemocyte and plasma proteins were separated by two‐dimensional electrophoresis; spot patterns were qualitatively compared between treatments within each experiment and the spots indicating differential protein expression associated with P. olseni challenge or with field infection were processed for protein identification. Fifteen clam proteins (four in haemocytes and eleven in plasma) of which expression was markedly affected by P. olseni were identified. Some of the identified proteins have a well‐known role in clam immune response against the parasite, such as lysozyme and lectins. Rho GTPase‐activating protein 6 could be a marker of resistance against P. olseni, which should be further studied.     

菲律宾蛤仔高密度育苗技术

设计和制造了可以大规模应用于菲律宾蛤仔（Ruditapes philippinarum）等经济贝类苗种生产的高密度幼体培育系统、高密度稚贝培养系统,并建立了与之相配套的贝类育苗新工艺。克服了传统育苗方式占地面积大、生产效率低、种苗质量和产量不稳定的弱点,具有低消耗、高质量和高效益的突出特点,是贝类育苗工艺的变革。利用这一育苗技术,可以充分发挥高密度、集约化生产的优势,通过对苗种培育过程中水质、饵料和环境条件的全面控制,消除或减少敌害生物的影响,提高幼体的抗逆能力,从而杜绝抗生素的使用;在降低育苗生产的环境污染同时,向养殖户提供健康、优质的贝类苗种,是一种与环境相协调的、可持续的水产苗种培育模式。     

Growth,mortality and reproduction of the transplanted Manila clam (Ruditapes philippinarum Adams & Reeve 1850) in Jiaozhou Bay

Samples of Manila clam (Ruditapes philippinarum Adams & Reeve 1850) were collected from May 2004 to April 2005 monthly, and plankton net trawling of planktonic larvae and bottom sediment sampling surveys were further conducted from May to October 2006 in Jiaozhou Bay. Based on the data collected, growth, mortality and reproduction of the transplanted Manila clam and the environmental effects were examined. The results showed that the enhanced clams grew well and showed a growth trend similar to the local wild ones. The main growth periods lasted from April to September, with the water temperature being the main factor affecting the growth, which was the same as that of the wild clams. There were also two reproduction cycles for the farmed Manila clams each year in Jiaozhou Bay and the main breeding period was from May to June. The phenomenon of delayed metamorphosis was quite common through larval development. The farmed clams could spawn when they reached sexual maturity, but they could not perform effective recruitment as many planktonic larvae died during metamorphosis and settlement. A preliminary study indicated that sediment perturbation and marine environment pollution were the main factors causing the death of larvae in the development process.     

基于生态系统动力学模型的胶州湾菲律宾蛤仔养殖容量动态评估

养殖容量评估是衡量贝类养殖活动是否环境友好、碳汇功能能否充分发挥的重要前提。本研究基于2018年5月—2019年2月的走航观测和定点连续观测数据,通过构建营养盐–浮游植物–浮游动物–碎屑–菲律宾蛤仔(nutrients–photoplankton–zooplankton–detritus–clams, NPZD-C)生态系统动力学模型,动态评估了胶州湾菲律宾蛤仔(Ruditapes philippinarum)的养殖容量。结果显示,构建的生态系统动力学模型能够较好地反演菲律宾蛤仔的生长和浮游植物的动态响应,菲律宾蛤仔和浮游植物的实测值和模拟值均呈显著线性相关(P<0.01),R2分别为0.934 8和0.926 4;不同放苗密度情境下的产量模拟结果显示,当苗种(2000~3000 ind./kg)的初始放苗密度分别为300、500、700、1000、1500 ind./m2时,蛤仔的预测产量分别为10.5、15.6、18.9、21.6、23.2 t/hm2;养殖容量评估结果显示,若在期望的10个月养殖时间内收获湿重为5 g以上的商品蛤仔,放苗密度需控制在1000 ind./m2以内,以生态效益和经济效益的最大化为判定标准,适宜的放苗密度为550~750 ind./m2。研究结果可为实施生态系统水平的胶州湾菲律宾蛤仔养殖管理、充分发挥菲律宾蛤仔的碳汇功能提供理论依据和科学指导。     

不同地理群体菲律宾蛤仔壳形态对体重性状的影响研究

采集我国南北沿海9个地理群体的菲律宾蛤仔(Ruditapes philippinarum),利用通径分析等方法评估各群体形态性状对活体重和软体重的影响。结果显示,各群体壳长、壳高、壳宽和壳厚4 个形态性状对菲律宾蛤仔活体重和软体重的影响存在显著差异。除壳厚外,其他形态性状与活体重和软体重呈显著相关(P < 0.05)。通径分析和决定系数分析显示,大多数群体的壳宽对活体重和软体重的直接作用最大。2个形态比例参数(壳宽/壳长和壳高/壳长)最大值出现在山东莱州群体(0.49和0.74),而最小值出现在大连东港群体(0.42和0.67)。壳厚的变异系数最大(22.74),而壳高的变异系数最小(9.47)。通过检验偏回归系数的显著性,建立了各群体形态性状对软体重的最优回归方程。聚类分析表明,菲律宾蛤仔不同群体未出现明显的地域分布特征,而呈现出不规律的南北交替聚类现象。本研究查明了不同地理群体菲律宾蛤仔壳形态性状对体重性状的影响,为菲律宾蛤仔地理群体的形态判别、种质资源分析和遗传育种研究等提供了重要科学依据。     

An enzyme-linked immunosorbent assay (ELISA) for detecting penaeid baculovirus

Abstract. A technique for detecting shrimp haculovirus based upon the sandwich indirect enzyme-linked immunosorbent assay ( elisa ) was developed. Baculovirus antiserums were developed in rabbits and guinea-pigs. Wells of microagglutination plates were coated with guinea-pig antiserum. Various dilutions of the purified virus preparation suspended either in Hanks' balanced salt solution (BSS) or in 30% hepatopancreas tissue in BSS were placed in wells. After washing, secondary anti-serum (rabbit antibaculovirus serum) was added. Goat horseradish peroxidase-labelled anti-rabbit globulin was used to detect reactions between baculovirus and rabbit antiserum. At least 10 ng viral protein could be detected by the technique.     

不同地理群体菲律宾蛤仔的选择反应及现实遗传力

对不同地理群体的3龄菲律宾蛤仔(Pp莆田群体、Dp大连群体、Tp东京群体)进行了混合选择。测量了各实验组的壳长,计算了不同地理群体菲律宾蛤仔的选择反应和现实遗传力。结果表明,3个地群体菲律宾蛤仔子代的上选组壳长显著大于对照组(P<0.05)。在不同生长发育阶段,菲律宾蛤仔的选择反应(R)和现实遗传力(hR2)随着日龄的增大而减小,即R幼虫培育期0.804±0.084>稚贝期0.705±0.039>养成期0.671±0.024;hR2幼虫期0.458±0.051>稚贝期0.402±0.025>养成期0.382±0.013。从总体水平上分析,菲律宾蛤仔R为0.726±0.1074,莆田群体、大连群体、东京群体的R分别为0.758±0.101、0.690±0.049、0.732±0.059;hR2为0.414±0.044,莆田群体、大连群体、东京群体的hR2分别为0.432±0.058、0.393±0.028、0.417±0.033。地理群体间的R和hR2次序为莆田群体>东京群体>大连群体,且彼此间无显著差异。     

家系内大、小两种规格菲律宾蛤仔的双列杂交

以壳长为标准,在菲律宾蛤仔生长速度快的家系中选择大、小两种规格蛤仔,上选雌性个体为A、雄性为B;下选雌性个体为a、雄性为b,采用双列杂交方法,分别建立AB、Ab、aB、ab4组近交家系。测量并统计分析各近交家系的幼虫期和稚贝期的壳长生长及变态情况。结果表明:近交家系的生长顺序为AB>Ab>aB>ab,除9日龄外,AB与ab的壳长生长差异显著(P<0.05)。随着日龄的增加,AB逐渐体现出明显的生长优势,在90日龄时与其它三个家系的生长差异明显(P<0.05)。杂交组Ab的生长优于aB,表明菲律宾蛤仔前期的生长也受母本效应的影响。从6日龄起,各近交家系开始附着变态,AB的变态率为71.12%1.53%,与Ab、aB差异不显著(P>0.05),与ab(41.6%1.33%)差异显著(P<0.05)。家系内近交改变了蛤仔附着变态时期的壳长生长分布频率,上选组AB壳长分布趋于大型化,而下选组ab壳长分布趋于小型化,Ab,aB两家系近似正态分布。研究表明,在家系内上选生长性状优良个体进行逐代选育是培育蛤仔速生新品种的有效手段。     

迟缓爱德华氏菌间接ELISA快速检测法

以迟缓爱德华氏菌(Edwardsiella tarda)WY28作为抗原,免疫新西兰兔,获得效价为1:2 048的多克隆抗体;以此多克隆抗体作为-抗,山羊抗兔IgG-HRP作为酶标二抗,建立迟缓爱德华氏菌的间接EIJSA快速检测法.采用棋盘滴定法确定抗原与一抗的最佳工作浓度分别为106 CFU·mL-1和1:10 000;酶标二抗的最适工作浓度为1:1 000.病原菌检测灵敏度为每孔103 CFU.该方法标准化后具有快速、灵敏等特性,与肠杆菌科其他细菌参考菌株无交叉反应,具良好的特异性.对养殖场发病大菱鲆(Scophthalmus maximus)和半滑舌鳎中分离的细菌菌株进行检测,从56株分离菌株中检测出27株迟缓爱德华氏菌,阳性检出率为48.2%.该方法的建立有助于快速准确地诊断由迟缓爱德华氏菌引起的养殖鱼类病害.     

花蛤蒸煮液喷雾干燥法制蛤粉工艺优化研究

花蛤熟制加工过程中会产生大量加工副产物汤汁,为了将其开发成调味粉,对经酶解、浓缩后的花蛤蒸煮液进行喷雾干燥,在单因素试验的基础上进一步采用响应面法优化喷雾干燥热风温度、进料温度、总固形物含量等工艺参数。试验结果表明,花蛤蒸煮液制备蛤粉最佳的工艺条件为进料温度60℃、热风温度199℃、总固形物含量30%、雾化压为96MPa,该条件下蛤粉得率为64.2%,含水率为3.9%。     

环境因子对胶州湾移植底播菲律宾蛤仔滤水率的影响

在实验室条件下对胶州湾移植底播菲律宾蛤仔的滤水率进行了研究。结果表明,不同温度、盐度、pH值条件下的蛤仔滤水率差异显著;2、3龄蛤仔滤水率随环境因子的变化趋势呈极显著正相关。在设定的环境因子梯度下,蛤仔滤水率随着温度、盐度、pH值的升高呈现先增大、后减小的趋势。最大滤水率分别出现在温度23℃、盐度30、pH值8.2。     

菲律宾蛤仔对0~#柴油和南海流花原油的生物富集动力学特征

以菲律宾蛤仔(Ruditapes philippinarum)为实验对象,基于双箱动力学模型,研究了菲律宾蛤仔肌肉和内脏组织对0~#柴油和南海流花原油的生物富集特征。采用OgirinPro 8.5软件对富集结果进行非线性拟合,获得了2种油品的吸收速率常数(k_1)、释放速率常数(k_2)、生物富集系数(BCF)、理论平衡时石油烃质量分数(CAmax)和生物学半衰期(B1/2)等动力学参数。结果表明,菲律宾蛤仔对0~#柴油和南海流花原油的k_1分别为0.921~9.74和3.43~64.2,k_2分别为0.092 8~0.134和0.100~0.189,BCF分别为6.87~94.56和34.30~379.88,CAmax分别为17.18~67.62 mg·kg~(-1)和30.38~595.24 mg·kg~(-1),B_(1/2)分别为5.17~7.47 d和3.67~6.93 d。菲律宾蛤仔对0~#柴油和南海流花原油的k_1和BCF均随着水体中石油烃浓度的增大而减小,k_2和B1/2与水体中石油烃浓度无明显相关性,CAmax随着水体中石油烃浓度的增加而增大。通过比较菲律宾蛤仔2种组织对2种油品的生物富集过程,发现菲律宾蛤仔对南海流花原油的生物富集能力强于对0~#柴油的富集能力,而内脏对石油烃的生物富集能力高于肌肉。     

Gametogenesis and spawning in the carpet-shell clam, Ruditapes decussatus (L.) (Mollusca: Bivalvia), from the Atlantic coast of Morocco

Abstract. Aspects of gametogenesis of the carpet-shell clam, Ruditapes decussatus (L.), in two lagoons on the Atlantic coast of north Morocco were studied during December 1986 to February 1988. The clams sampled did not show any sign of hermaphroditism and the percentage of males was 42%. The gonads started developing in mid-winter and were ripe in spring. Successive spawnings and reconstitution of gametes look place simultaneously starting from May up to the end of September. Two major spawning periods were noticed: a partial one in May-June and a complete spawning in August-September. From October to December the gonads were at resting stages. In coastal ecosystems where freshwater input is a very important factor, gametogenesis may be affected if clams are exposed to low salinities (less than 15%) for prolonged periods.     

菲律宾蛤仔家系的建立及早期生长发育

采用不平衡巢式设计建立33个菲律宾蛤仔家系(11个父系半同胞家系和33个全同胞家系),并对各家系蛤仔的卵径、受精率、孵化率及生长、存活和变态的相关指标进行了分析。结果表明:各家系蛤仔的卵径、受精率无显著差异(P>0.05,n=90),但孵化率有明显差异(P<0.05,n=90)。在不同时期各家系蛤仔的生长情况不同。幼虫期间,9日龄C2生长最快比生长最慢的F2平均壳长大28.24%,且差异显著(P<0.05,n=90)。D3绝对生长最大比平均值高37.24%。稚贝期间,40日龄I1生长最快比生长最慢的B3平均壳长大78.29%,且差异显著(P<0.05,n=90)。I1绝对生长也最大比平均值高87.61%,其中400～500μm个体占30%,500μm以上个体占53.33%,家系内个体趋于大型化,而B1、B3家系内个体生长性状出现衰退现象,趋于小型化,300μm以下个体分别占整个家系的83.33%、90%。在相同时期各家系蛤仔的存活率不同。幼虫期间,9日龄I2存活率最高比平均存活率高94.14%,E3存活率最低比平均存活率低72.65%。稚贝期间,40日龄时各家系稚贝的存活率较高,都在85%以上。变态期间,同胞...     

Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

罗非鱼源无乳链球菌的间接ELISA快速检测方法

以罗非鱼源无乳链球菌为抗原,免疫新西兰兔获得高效价的多克隆抗体;以此多克隆抗体作为一抗,羊抗兔Ig G-HRP作为酶标二抗,建立无乳链球菌的间接ELISA快速检测法。采用棋盘滴定法确定抗原与一抗的最佳工作浓度分别为106 CFU/m L和1∶10 000;酶标二抗的最适工作浓度为1∶1000。病原菌检测灵敏度为每孔103 CFU。该方法标准化后具有快速、灵敏等特性,与海豚链球菌等其他常见水产病原菌无交叉反应;阻断实验中的阻断率达72.02%;交叉反应和阻断实验的结果显示该方法具有较高的特异性。将该方法标准化后检测了44株2007—2013年分离的罗非鱼无乳链球菌,阳性检测率为100%;对人工感染后死亡的30尾罗非鱼的脑组织进行检测,阳性检测率为100%;对人工感染后未死亡的20尾罗非鱼的脑,肝和脾组织进行检测,脑的阳性检测率为0%,肝的阳性检测率为25%,脾的阳性检测率为50%;表明该技术不仅能够检测已发病的罗非鱼,而且能够检测无症状的带菌罗非鱼,该方法的建立有助于快速准确地诊断由无乳链球菌引起的罗非鱼链球菌病。     

Effects of temperature and salinity on oxygen consumption and ammonia excretion in different colour strains of the Manila clam,Ruditapes philippinarum

The metabolic responses of different colour strains of Ruditapes philippinarum in terms of oxygen consumption and ammonia excretion to changes in temperature (15–35°C) and salinity (20–40) were investigated. In our range of temperatures (15–35°C), oxygen consumption rate (OCR) increases in cultivated strains (White and Zebra) in opposition to the effect in the wild strain which reach a maximum at 25°C. The highest Q₁₀ coefficients were 2.741 for zebra strain, 4.326 for white strain, and 1.944 for wild at temperatures of 25–30, 30–35 and 20–25°C respectively. In our range of salinity (20–40°C), OCRs of white strain and zebra strain firstly decreased to lowest level at 25 and 30, and then increased to highest level at 35 and 40 respectively. When the salinity is beyond 35, the OCR decreased and the turning point was found in the white strain and wild, but the zebra strain OCR still increased to a highest level (1.906 mg g^?1 h^?1) at 40 (P < 0.05). These results show that the cultivated colour strains of R. philippinarum were different from wild in terms of metabolic responses, and information on its response to different temperature and salinity have implications in the aquaculture industry.     

Metabolic characteristics of the Japanese clam Ruditapes philippinarum (Adams & Reeve) during aerial exposure

Some metabolic end‐products in tissues and physiological parameters of haemolymph of the Japanese clam Ruditapes philippinarum (Adams & Reeve) were investigated under aerial exposure at 15 °C with low relative humidity (RH) (22%) or high RH (91%). Acclimated clams in water at 15 °C were used as a control and to supply the low or high RH experiment. The low RH experiment was conducted at air exposure for 50 h (LH₅₀), and the high RH experiment for 50 h (HH₅₀) and 100 h (HH₁₀₀). Accumulation of succinate and alanine in the tissues proved that the clam shifted its metabolism to anaerobiosis. Lactate accumulation did not occur. Glucose concentrations in tissues increased slightly during exposure except for HH₅₀. The total free amino acid contents (363–410 μmol g^?1) were higher than those of glucose (530–804 μmol 100 g^?1), suggesting that the free amino acids might play a more dominant role in the metabolism. Increase in the haemolymph pco ₂ did not influence the acid–base balance in the haemolymph during exposure at both humidity conditions. Increase in the haemolymph ammonia from 48.2 to 57.1 μmol 100 g^?1 and to 131.0 μmol 100 g^?1 at LH₅₀ and HH₁₀₀, respectively, suggested that ammonia might function as an important buffering factor during aerial exposure. Retention values of the haemolymph po ₂ even at the longest exposure time (16.30 mmHg at LH₅₀ and 14.69 mmHg at HH₁₀₀) indicated that the clams depend partially on some aerobic manner.